Target Detection with Nanopore

ABSTRACT

Provided are methods for detecting a target molecule or particle suspected to be present in a sample, comprising (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore comprising an opening in a structure that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device further comprises a sensor configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule or particle bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT Application No. PCT/US2014/046397, filed Jul. 11, 2014, which claims the benefit of PCT Application No. PCT/US2014/036861, filed May 5, 2014, the contents of which are each incorporated by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 20, 2016, is named 34004US_CRF_sequencelisting.txt and is 563 bytes in size.

BACKGROUND

Detection of nano-scale and micro-scale particles, such as circulating tumor cells, bacteria and viruses, has immense clinical utility. Currently available methods include immunohistochemistry and nucleic acid-based detection, and cell proliferation is typically required before a sensitive detection can be carried out.

Molecular detection and quantitation are also important, and can be carried out with various methods depending on the type of the molecule. For instance, a nucleotide sequence can be detected by virtue of its sequence complementarity to a probe or primer, through hybridization and/or amplification, or in fewer occasions, with a protein that recognizes the sequence. A protein, on the other hand, is commonly detected with an antibody that specifically recognizes and binds the protein. An enzyme-linked immuno sorbent assay (ELISA), in this respect, is highly commercialized and commonly used.

Methods also exist for detecting or quantitating various other large or small molecules, such as carbohydrates, chemical compounds, ions, and elements.

Methods and systems for highly sensitive detection of molecules as well as particles, such as tumor cells and pathogenic organisms, have broad applications, in particular, clinically, for pathogen detection and disease diagnosis, for instance. Additionally, such detection may: allow for the personalization of medical treatments and health programs; facilitate the search for effective pharmaceutical drug compounds and biotherapeutics; and enable clinicians to identify abnormal hormones, ions, proteins, or other molecules produced by a patient's body and/or identify the presence of poisons, illegal drugs, or other harmful chemicals ingested or injected into a patient.

Currently available techniques for the detection of molecules and particles are generally expensive, labor-intensive, skill-intensive, and/or time-intensive. A need exists for improved detection techniques, which produce accurate results quickly, cheaply, and easily.

SUMMARY

Various aspects disclosed herein may fulfill one or more of the above-mentioned needs. The systems and methods described herein each have several aspects, no single one of which is solely responsible for its desirable attributes. Without limiting the scope of this disclosure as expressed by the claims that follow, the more prominent features will now be discussed briefly. After considering this discussion, and particularly after reading the section entitled “Detailed Description,” one will understand how the sample features described herein provide for improved systems and methods.

In one embodiment, the present disclosure provides a method for assaying whether a target molecule or particle is present in a sample, the method comprising: (a) contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain of the fusion molecule is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; (b) loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device comprises a sensor configured to identify objects passing through the pore; and (c) determining, with the sensor, whether the fusion molecule bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence or absence of the target molecule or particle in the sample.

In some aspects, the target molecule is selected from the group consisting of a protein, a peptide, a nucleic acid, a chemical compound, a lipid, a receptor, an ion, and an element.

In some aspects, the target particle is selected from the group consisting of protein complexes and protein aggregates, peptide aggregates, protein/nucleic acid complexes, fragmented or fully assembled viruses, bacteria, cells, and cellular aggregates.

In some aspects, step (a) of the method for assaying whether a target molecule or particle is present in a sample is performed prior to step (b). In some aspects, step (b) is performed prior to step (a).

In some aspects, the method further comprises applying a condition suspected to alter the binding between the target molecule or particle and the ligand, and carrying out the determination again. In some aspects, the condition is selected from the group consisting of removing the target molecule or particle from the sample, adding an agent that competes with the target molecule or particle or the ligand for binding, and changing the pH, salt concentration, or temperature.

In some aspects, the binding motif comprises a chemical modification for binding to the binding domain. In some aspects, the chemical modification is selected from the group consisting of acetylation, methylation, summolation, glycosylation, phosphorylation, and oxidation.

In some aspects, the polymer comprises a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), a DNA/RNA hybrid, or a polypeptide. In some aspects, the polymer is a synthetic scaffold.

In some aspects, the binding domain is selected from the group consisting of a helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix turn helix, a helix-loop-helix and an HMG-box.

In some aspects, the binding domain is selected from the group consisting of locked nucleic acids (LNAs), PNAs, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), peptides, dendrimers, and aptamers (DNA and/or protein).

In some aspects, the ligand is a protein. In some aspects, the ligand is selected from the group consisting of an antibody, an antibody fragment, an epitope, a hormone, a neurotransmitter, a cytokine, a growth factor, a cell recognition molecule, a nucleic acid, a peptide, and a receptor. In some aspects, the ligand is an aptamer (e.g., DNA, protein, or DNA/protein). In some aspects, the ligand is a small molecule compound.

In some aspects, the binding domain and the ligand are linked via a covalent bond, a hydrogen bond, an ionic bond, a metallic bond, van der Walls force, a hydrophobic interaction, or a planar stacking interaction, or are translated as a continuous polypeptide, to form the fusion molecule.

In some aspects, the method further comprises contacting the sample with a detectable label capable of binding to the target molecule, target particle or target/ligand complex.

In some aspects, the polymer comprises at least two units of the binding motif.

In some aspects, the polymer comprises at least two different binding motifs. In such aspects, the sample is in contact with at least two fusion molecules, each of which comprises a different binding domain capable of binding to a different one of the at least two different binding motifs, and a different ligand capable of binding to a different target molecule or particle; and the sensor is configured to identify whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.

In some aspects, the sensor comprises electrodes further configured to apply a voltage across the two volumes.

In some aspects, the device comprises an upper chamber, a middle chamber and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore.

In one aspect, the first pore and second pore are about 1 nm to about 100 nm in diameter. Such pores can be suitable for detecting molecules such as proteins and nucleic acids. In one aspect, the first pore and second pore are as large as about 50,000 nm in diameter, which can be suitable for detecting larger particles such as tumor and bacterial cells.

In some aspects, the pores are about 10 nm to about 1000 nm apart from each other. In some such aspects, the distance between the pores is sized such that the polymer scaffold may simultaneously extend through both the first and second pores. In other aspects, the pores are more than 1000 nm apart from each other.

In some aspects, each of the chambers comprises an electrode for connecting to a power supply.

In some aspects, the method further comprises moving the polymer in a reverse direction after the binding motif passes through at least one pore, such as to identify, again, whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.

Also provided are kits, packages or mixtures that detect the presence of a target molecule or particle. In some aspects, the kit, package or mixture is comprised of (a) a fusion molecule, which itself is a ligand capable of binding to the target molecule or particle and a binding domain, (b) a polymer scaffold, which is comprised of at least one binding motif to which the binding domain is capable of binding, (c) a device, which is comprised of a pore that separates an interior space of the device into two volumes, wherein the device is configured to allow the polymer to pass through the pore from one volume to the other volume, and wherein the device is further comprised of a sensor configured to identify whether the binding motif is (i) bound to the fusion molecule while the ligand is bound to the target molecule or particle, (ii) bound to the fusion molecule while the ligand is not bound to the target molecule or particle, or (iii) not bound to the fusion molecule. In some aspects, the device is further comprised of a second pore that further separates the interior space of the device such that three volumes: an upper chamber, a middle chamber, and a lower chamber, are present.

In some aspects, the kit, package or mixture further comprises a sample suspected of containing the target molecule or particle. In some aspects, the sample further comprises a detectable label capable of binding to the target molecule, particle, ligand/target complex, or ligand/particle complex.

BRIEF DESCRIPTION OF THE DRAWINGS

Provided as embodiments of this disclosure are drawings that illustrate features by exemplification only, and not limitation.

FIG. 1 illustrates the detection of a target molecule or particle with one embodiment of the presently disclosed method.

FIG. 2 provides the illustration of a more specific example, where a double-stranded DNA is used as the polymer scaffold, and a human immunodeficiency virus (HIV) envelope protein is used as the ligand. The combination is used to detect an anti-HIV antibody.

FIG. 3A, FIG. 3B, and FIG. 3C show representative and idealized current profiles of three example molecules, demonstrating that binding between a target molecule (or particle) and a fusion molecule can be detected when passing through a nanopore, since it has a different current profile, compared to that of the fusion molecule alone or the DNA alone. Specifically, FIG. 3A shows current profiles consistent with higher salt concentrations (>0.4 M KCl, for example at 1M KCl) in the experimental buffer and a positive applied voltage, generating a positive current flow through the pore. By another example, FIG. 3B shows current profiles consistent with lower salt concentrations (>0.4 M KCl, for example at 100 mM KCl) in the experimental buffer and again at a positive applied voltage. By another example, FIG. 3C shows current profiles consistent with lower salt concentrations (>0.4 M KCl, for example at 100 mM KCl) in the experimental buffer and a negative applied voltage.

FIG. 4 illustrates the multiplexing capability of the present technology by including different binding motifs in the polymer scaffold. Such multiplexing can be accomplished with one nanopore or more than one nanopore.

FIG. 5A, FIG. 5B, and FIG. 5C illustrate a nanopore device with at least two pores separating multiple chambers.

Specifically, FIG. 5A is a schematic of a dual-pore chip and a dual-amplifier electronics configuration for independent voltage control (V₁ or V₂) and current measurement (l₁, or l₂) of each pore. Three chambers, A-C, are shown and are volumetrically separated except by common pores.

FIG. 5B is a schematic where electrically, V₁ and V₂ are principally applied across the resistance of each nanopore by constructing a device that minimizes all access resistances to effectively decouple l₂ and l₂.

FIG. 5C depicts a schematic in which competing voltages are used for control, with arrows showing the direction of each voltage force.

FIGS. 6A, 6B, and 6C illustrate a nanopore device having one pore connecting two chambers and example results from its use. Specifically, FIG. 6A depicts a schematic diagram of the nanopore device. FIG. 6B depicts a representative current trace showing a blockade event resulting from the passage of a double-stranded DNA passing through the pore. The current amplitude shift amount (Δl=l₀−l_(B)) and duration to are used to quantify the passage event. FIG. 6C depicts a scatter plot showing the change in current amount (Δl) vs. translocation time (t_(D)) for all blockade events recorded over 16 minutes.

FIG. 7A and FIG. 7B depict current traces measured within one embodiment of a nanopore device fabricated in accordance with the present invention. The provided current traces show that unbound dsDNA causes current enhancement events at KCl concentrations below 0.4 M. Current enhancements appeared as downward shifts in the provided experiment, since the voltage and current are both negative (as in FIG. 3C). Specifically, in DNA alone control experiments using a 10-11 nm diameter pore in 0.1M KCl at −200 mV, 5.6 kb dsDNA scaffold (FIG. 7A) causes brief current enhancement events that are 50-70 pA in amplitude and 10-200 microseconds in duration. Likewise, 48 kb Lambda DNA (FIG. 7B) causes current enhancement events 50-70 pA in amplitude and 50-2000 microseconds in duration.

FIG. 8 depicts a schematic diagram of a polymer scaffold. Specifically, FIG. 8 shows a 5,631 by dsDNA scaffold and the location of 10 total VspR binding sites. Of the 10 VspR binding sites, 5 are of one 14 base-pair sequence, 3 of a different 18 base pair sequence, and 2 are of a 27 base pair sequence. Also shown are the distances (in base pairs) between the binding sites.

FIG. 9A and FIG. 9B each show schematic representations of embodiments of a nanopore with a scaffold passing therethrough. Each also shows a resultant current profile associated with the scaffold passage as measured by one embodiment of the disclosed nanopore device. In particular, FIG. 9A and FIG. 9B compare events with DNA scaffold alone (FIG. 9A) and VspR-bound DNA (FIG. 9B). Specifically, FIG. 9A shows a graphic depicting the 5,631 by dsDNA scaffold passing through the pore, and a representative current enhancement event (downward 50 pA shift lasting 100 microseconds) when the scaffold passes through the pore. FIG. 9B shows a graphic depicting multiple VspR bound to a dsDNA scaffold that is passing through the pore, and a representative current attenuation event (upward 150 pA shift lasting 1.1 milliseconds) when the VspR-bound scaffold passes through the pore. At an applied voltage of −100 mV, the open channel current is negative, so downward events correspond to current enhancement events, and upward events correspond to current attenuation events (as in FIG. 3C). The shift direction is preserved, even though the baseline is zeroed for display purposes.

FIG. 10 shows ten more representative current attenuation events depicted in a current profile consistent with the VspR-bound scaffold passing through the pore. All shifts are consistent with current attenuations; the baseline is zeroed for display purposes.

FIG. 11 shows two representative current events depicted in a current profile captured in an experiment with 5.6 kb dsDNA scaffold and RecA protein at 180 mV and 1M KCl using a 16-18 nm diameter nanopore. The first event is consistent with an unbound dsDNA or possibly a free RecA (or multiple associated RecA proteins) passing through the pore, at 280 pA mean current attenuation lasting 70 microseconds. The second event is consistent with RecA-bound scaffold passing through the pore, at 1.1 nA mean current attenuation lasting 2.7 milliseconds. RecA-bound events commonly display deeper blockades with longer duration.

FIG. 12 depicts four more current profiles, each showing a representative current event consistent with RecA-bound scaffold passing through the pore.

FIG. 13 shows scatter plots and histograms depicting all 1385 events recorded over 10 minutes in one experiment conducted using embodiments of methods described herein. In the depicted graphs, one data point is provided for each event. In particular, the depicted graphs show: (a) maximum conductance in nS (maximum current shift in pA divided by voltage in mV) vs. time duration in seconds, with time duration on a log-scale; (b) a probability histogram of the maximum conductance shift values; (c) mean conductance (mean current shift divided by voltage) vs. time duration, with time duration on a log-scale; (d) a probability histogram of the mean conductance values; and (e) a probability histogram of the time duration on a log-scale.

FIG. 14A, FIG. 14B, and FIG. 14C illustrate results from a nanopore device detecting DNA/RecA complexes and RecA-antibody on DNA/RecA complexes, and the results differentiating these complexes from unbound DNA and also from free RecA.

Specifically, FIG. 14A is a gel shift assay. Specifically, the DNA/RecA/mAb ARM191 Gel Shift Experiments (EMSA) have lanes: 1) Ladder, top rung 5000 bp; 2) Scaffold DNA only in RecA labeling buffer; 3) DNA/RecA complex, 1:1 RecA protein to theoretical RecA binding sites; 4) DNA/RecA/Ab complex, DNA/Rec incubated with a 1:2000 dilution of monoclonal Ab ARM191; 5) Scaffold DNA only in Ab labeling buffer; and 6) Scaffold DNA mixed with mAb (ARM191).

FIG. 14B shows representative events for DNA (230 pA, 0.1 ms), DNA/RecA (390 pA, 1.1 ms), and probable DNA/RecA/Ab (860 pA, 1.5 ms). RecA-bound DNA event amplitudes are uniformly smaller than in earlier figures (FIGS. 11-13) since the pore used to measure these events is considerably larger (27-29 nm in diameter).

FIG. 14C depicts a (i) Scatter plot of lΔ/l vs. t_(D) and (ii) horizontal probability histogram of lΔ/l for two separate experiments overlaid. In a RecA alone control experiment, 0.5 uM RecA (*) was measured at 180 mV in 1M KCl with a 20 nm diameter pore, generating 767 events over 10 min. Note that only 0.6% of RecA events exceed a criteria of (600 pA, 0.2 ms) under these conditions. In another experiment, three reagents were added in sequence in 1M LiCl. First, 0.1 uM DNA (□) was measured at 200 mV with a 20 nm diameter pore, generating 402 events at 0.1 events/sec. After the pore enlarged to 27 nm, 1.25 nM DNA/RecA (⋅) was added, generating 3387 events at 1.44 events/sec. Lastly, 1.25 nM DNA/RecA/Ab (◯) was added generating 4953 events at 4.49 events/sec. Events exceeding the (600 pA, 0.2 ms) criteria grew monotonically from 0% with DNA alone, to 5.2% (176) with DNA/RecA added, and up to 9.8% (485) with DNA/RecA/Ab added. While RecA could have increased event durations in LiCl, as shown for DNA, event amplitudes are unlikely to shift significantly toward the (600 pA, 0.2 ms) criteria.

FIG. 15A and FIG. 15B illustrates schematic diagrams of polymer scaffolds consistent with embodiments of the present disclosure. Specifically, FIG. 15A shows a 5.6 kb dsDNA scaffold used in various experiments, the scaffold having been engineered to bind 12-mer peptide-nucleic-acid (PNA) molecules, with each PNA having 3 biotinylated sites for binding avidin (e.g., neutravidin, and or monovalent streptavidin). Also shown is FIG. 15B identifying the 25 distinct PNA binding sites on the scaffold that localize up to 75 avidin biomarker binding sites.

FIG. 16A, FIG. 16B, FIG. 16C, and FIG. 16D illustrate: (FIG. 16A) a schematic of the 5.6 kb dsDNA scaffold passing through a nanopore; (FIG. 16B) a schematic of a free neutravidin passing through a nanopore; (FIG. 16C) a schematic of the dsDNA labeled with a PNA passing through a nanopore, the PNA having all three biotin sites bound by Neutravidin; and (FIG. 16D) corresponding current traces as measured in the chamber above the pore in a nanopore device fabricated in accordance with the present disclosure. In FIG. 16D, the current traces depict representative translocation events in the recorded current from separate nanopore experiments with DNA alone, Neutravidin alone, and DNA/PNA/Neutravidin complexes. As detailed in the examples, the deeper and longer event pattern in the D/P/N experiment is identified as a DNA/PNA/Neutravidin event and is clearly distinguished from DNA alone or Neutravidin alone events.

FIG. 17A, FIG. 17B, and FIG. 17C illustrate: (FIG. 17A) a scatter plot of the current shift vs. the duration (lΔ/l vs. t_(D)) of all recorded events for three separate experiments at 200 mV with 10

11 nm diameter pores, the experiments including: (D)—5.6 kb dsDNA alone at 1 nM yielding 1301 events over 16 minutes; (N) Neutravidin alone at 80 nM yielding 2589 events over 11 minutes; and (D/P/N) DNA/PNA/Neut complexes at 60 pM yielding 4198 events over 7.3 minutes. D/P/N subpopulations overlap with the N and D control experiment populations, with most events in the DPN experiment matching unbound N event characteristics; (FIG. 17B) a horizontal probability histogram of lΔ/l for the three data sets. The inset shows a histogram for a subset of 578 DPN events with t_(D)>0.08 ms, which attempts to trim out non-DNA events from the D/P/N data set (from controls, 8% of N events and 54% of D events have to >0.08 ms). Such DPN events have significant spread in lΔ/l, with 252 (6% of the total) of these longer-duration events above 2.4 nA, whereas from controls, only 18 (0.7%) N events and 33 (2.5%) D events have (lΔ/l, t_(D))>(2.4 nA, 0.08 ms); and (FIG. 17C) DNA/PNA/Neutravidin Gel Shift Experiments (EMSA) with lanes: 1) sizing Ladder, top rung 5000 bp; 2) DNA only in labeling buffer; 3) DNA/PNA+3× excess Neut to biotin; 4) DNA/PNA+7× excess Neut to biotin; 5) DNA/PNA+16× excess Neut to biotin; 6) DNA/PNA+36× excess Neut to biotin; and 7) DNA/PNA in labeling buffer.

Some or all of the figures are schematic representations for exemplification; hence, they do not necessarily depict the actual relative sizes or locations of the elements shown. The figures are presented for the purpose of illustrating one or more embodiments with the explicit understanding that they will not be used to limit the scope or the meaning of the claims that follow below.

DETAILED DESCRIPTION

Throughout this application, the text refers to various embodiments of the present devices, compositions, systems, and methods. The various embodiments described are meant to provide a variety of illustrative examples and should not be construed as descriptions of alternative species. Rather, it should be noted that the descriptions of various embodiments provided herein may be of overlapping scope. The embodiments discussed herein are merely illustrative and are not meant to limit the scope of the present invention.

Also throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure in their entireties.

As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an electrode” includes a plurality of electrodes, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that the systems, devices, and methods include the recited components or steps, but not excluding others. “Consisting essentially of” when used to define systems, devices, and methods, shall mean excluding other components or steps of any essential significance to the combination. “Consisting of” shall mean excluding other components or steps. Embodiments defined by each of these transition terms are within the scope of this invention.

All numerical designations, e.g., distance, size, temperature, time, voltage and concentration, including ranges, are approximations which are varied (+) or (−) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. It also is to be understood, although not always explicitly stated, that the components described herein are merely exemplary, and that equivalents of such are known in the art.

As used herein, “a device comprising a pore that separates an interior space” shall refer to a device having a pore that comprises an opening within a structure, the structure separating an interior space into more than one volume or chamber.

Molecular Detection

The present disclosure provides methods and systems for molecular detection and quantitation. In addition, the methods and systems can also be configured to measure the affinity of a molecule binding with another molecule. Further, such detection, quantitation, and measurement can be carried out in a multiplexed manner, greatly increasing its efficiency.

FIG. 1 provides an illustration of one embodiment of the disclosed methods and systems. More specifically, the system includes a ligand 104 that is capable of binding to a target molecule 105 to be detected or quantitated. The ligand 104 can be part of, or be linked to, a binding moiety (referred to as “binding domain”) 103 that is capable of binding to a specific binding motif 101 on a polymer scaffold 109. Together, the ligand 104 and the binding domain 103 form a fusion molecule 102. In various embodiments, both components of the fusion molecule 102 (i.e., both the ligand 104 and the binding domain 103) bind to their respective targets (e.g., target molecule 105 and binding motif 101, respectively) with high affinity and specificity.

Therefore, if all are present in a solution, the fusion molecule 102 binds, on one end, to a polymer scaffold (or simply, “polymer”) 109 through the specific recognition and binding between the binding motif 101 and the binding domain 103, and on the other end, to the target molecule 105 by virtue of the interaction between the ligand 104 and the target molecule 105. Such bindings cause the formation of a complex that includes the polymer 109, the fusion molecule 102 and the target molecule 105.

The formed complex can be detected using a that includes a nanopore (or simply, pore) 107, and a sensor. The pore 107 is a nano-scale or micro-scale opening in a structure separating two volumes. The sensor 107 may be positioned within or adjacent the pore 107 or elsewhere within the two volumes. The sensor is configured to identify objects passing through the pore 107. For example, in some embodiments, the sensor identifies objects passing through the pore 107 by detecting a change in a measurable parameter, wherein the change is indicative of an object passing through the pore 107. This device is referred throughout as a “nanopore device”. In some embodiments, the nanopore device 108 includes means, such as electrodes connected to power sources, for moving the polymer 109 from one volume to another, across the pore 107. As the polymer 109 can be charged or be modified to contain charges, one example of such means generates a potential or voltage across the pore 107 to facilitate and control the movement of the polymer 109. In a preferred embodiment, the sensor comprises a pair of electrodes, which are configured to both detect the passage of objects, and provide a voltage, across the pore 107. In this embodiment, a voltage-clamp or a patch-clamp is used to simultaneously supply a voltage across the pore and measure the current through the pore.

When a sample that includes the formed complex is loaded in the nanopore device 108, the nanopore device 108 can be configured to pass the polymer 109 through the pore 107. When the binding motif 101 is within the pore or adjacent to the pore 107, the binding status of the motif 101 can be detected by the sensor.

The “binding status” of a binding motif, as used herein, refers to whether the binding motif is bound to a fusion molecule with a corresponding binding domain, and whether the fusion molecule is also bound to a target molecule. Essentially, the binding status can be one of three potential statuses: (i) the binding motif is free and not bound to a fusion molecule (see 305 in FIG. 3A); (ii) the binding motif is bound to a fusion molecule that does not bind to a target molecule (see 306 in FIG. 3A); or (iii) the binding motif is bound to a fusion molecule that is bound to a target molecule (see 307 in FIG. 3A).

Detection of the binding status of a binding motif can be carried out by various methods. In one aspect, by virtue of the different sizes of the binding motif at each status, when the binding motif passes through the pore, the different sizes result in different electrical currents across the pore. In one aspect, as shown in FIG. 3A, with a positive voltage applied and KCl concentrations greater than 0.4 M in the experiment buffer, the measured current signals 301, when 305, 306, and 307 pass through the pore, are signals 302, 303, and 304, respectively. All three event types are subjected to current attenuation when KCl concentrations are greater than 0.4 M, causing a reduction in the positive current flow. The three signals 302, 303, and 304 can be differentiated from one another by the amount of the current shift (height) and/or the duration of the current shift (width), or by any other feature in the signal that differentiates the three event types. It may also be that 304 is commonly different than 302 and 303, but that 302 and 303 are not commonly different from each other, in which case, robust detection of the biomarker bound to the passing molecule can still be accomplished. In another aspect, as shown in FIG. 3B, with a positive voltage applied and KCl concentrations less than 0.4 M in the experiment buffer, the measured current signals 308, when 312, 313, and 314 pass through the pore, are signals 309, 310, and 311, respectively. Passage of dsDNA alone causes current enhancement events (309) at KCl concentrations less than 0.4 M. This was shown in the published research by Smeets, Ralph M M, et al. “Salt dependence of ion transport and DNA translocation through solid-state nanopores.” Nano Letters 6.1 (2006): 89-95. Hence, the signal 309 can be differentiated from 310 and 311 by the event amplitude direction (polarity) relative to the open channel baseline current level (308), in addition to the three signals commonly having different amounts of the current shift (height) and/or the duration of the current shift (width), or by any other feature in the signal that differentiates the three event types. In another aspect, as shown in FIG. 3C, with a negative voltage applied and KCl concentrations less than 0.4 M in the experiment buffer, the negative measured current signals 315, when 319, 320, and 321 pass through the pore, are signals 316, 317, and 318, respectively. Compared to signals 309, 310, and 311 with a positive voltage, the signals 316, 317, and 318 have the opposite polarity since the applied voltage has the opposite (negative) polarity. In all aspects of the FIG. 3A, FIG. 3B, and FIG. 3C embodiments, the sensor comprises electrodes, which are connected to power sources and can detect the current. Either one or both of the electrodes, therefore, serve as a “sensor.” In this embodiment, a voltage-clamp or a patch-clamp is used to simultaneously supply a voltage across the pore and measure the current through the pore.

In some aspects, an agent 106 as shown in FIG. 1 is added to the complex to aid detection. This agent is capable of binding to the target molecule or the ligand/target molecule complex. In one aspect, the agent includes a charge, either negative or positive, to facilitate detection. In another aspect, the agent adds size to facilitate detection. In another aspect, the agent includes a detectable label, such as a fluorophore.

In this context, an identification of status (iii) indicates that a polymer-fusion molecule-target molecule complex has formed. In other words, the target molecule is detected.

Particle Detection

The present disclosure also provides, in some aspects, methods and systems for detecting, quantitating, and measuring particles such as proteins, protein aggregates, oligomers, or protein/DNA complexes, or cells and microorganisms, including viruses, bacteria, and cellular aggregates.

In some aspects, the pore within the structure that separates the device into two volumes has a size that allows particles, such as viruses, bacteria, cells, or cellular aggregates, to pass through. A ligand that is capable of binding to a target particle to be detected or quantitated can be included in the solution in the nanopore device such that the ligand can bind to the unique target particle and the polymer scaffold through a binding domain and a binding motif to form a complex. Many such particles have unique markers on their surfaces that can be specifically recognized by a ligand. For instance, tumor cells can have tumor antigens expressed on the cell surface, and bacterial cells can have endotoxins attached on the cell membrane.

When the formed complex in a solution loaded into the nanopore device is moved along with the polymer scaffold to pass through the pore, the binding status of the complex within or adjacent to the pore can be detected such that the target microorganisms bound to the ligands can be identified using methods similar to the molecular detection methods described elsewhere in the disclosure.

Polymer Scaffold

A polymer scaffold suitable for use in the present technology is a scaffold that can be loaded into a nanopore device and passed through the pore from one end to the other.

Non-limiting examples of polymers include nucleic acids, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or peptide nucleic acid (PNA), dendrimers, and linearized proteins or peptides. In some aspects, the DNA or RNA can be single-stranded or double-stranded, or can be a DNA/RNA hybrid molecule.

In one aspect, the polymer is synthetic or chemically modified. Chemical modification can help to stabilize the polymer, add charges to the polymer to increase mobility, maintain linearity, or add or modify the binding specificity. In some aspects, the chemical modification is acetylation, methylation, summolation, oxidation, phosphorylation, glycosylation, or the addition of biotin.

In some aspects, the polymer is electrically charged. DNA, RNA, PNA and proteins are typically charged under physiological conditions. Such polymers can be further modified to increase or decrease the carried charge. Other polymers can be modified to introduce charges. Charges on the polymer can be useful for driving the polymer to pass through the pore of a nanopore device. For instance, a charged polymer can move across the pore by virtue of an application of voltage across the pore.

In some aspects, when charges are introduced to the polymer, the charges can be added at the ends of the polymer. In some aspects, the charges are spread over the polymer.

In one embodiment, each unit of the charged polymer is charged at the pH selected. In another embodiment, the charged polymer includes sufficient charged units to be pulled into and through the pore by electrostatic forces. For example, a peptide containing sufficient entities can be charged at a selected pH (lysine, aspartic acid, glutamic acid, etc.) so as to be used in the devices and methods described herein. Likewise, a co-polymer comprising methacrylic acid and ethylene is a charged polymer for the purposes of this invention if there is sufficient charged carboxylate groups of the methacrylic acid residue to be used in the devices and methods described herein. In one embodiment, the charged polymer includes one or more charged units at or close to one terminus of the polymer. In another embodiment, the charged polymer includes one or more charged units at or close to both termini of the polymer. One co-polymer example is a DNA wrapped around protein (e.g. DNA/nucleosome). Another example of a co-polymer is a linearized protein conjugated to DNA at the N- and C-terminus.

Binding Motifs and Binding Domains

For nucleic acids and polypeptides such as the polymer scaffold, a binding motif can be a nucleotide or peptide sequence that is recognizable by a binding domain. In some embodiments, the binding domain is a peptide sequence forming a functional portion of a protein, although the binding domain does not have to be a protein. For nucleic acids, for instance, there are proteins that specifically recognize and bind to sequences (motifs) such as promoters, enhancers, thymine-thymine dimers, and certain secondary structures such as bent nucleotide and sequences with single-strand breakage.

In some aspects, the binding motif includes a chemical modification that causes or facilitates recognition and binding by a binding domain. For example, methylated DNA sequences can be recognized by transcription factors, DNA methyltransferases or methylation repair enzymes. In other embodiments, biotin may be incorporated into, and recognized by, avidin family members. In such embodiments, biotin forms the binding motif and avidin or an avidin family member is the binding domain.

Molecules, in particular proteins, that are capable of specifically recognizing nucleotide binding motifs are known in the art. For instance, protein domains such as helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix turn helix, a helix-loop-helix and an HMG-box, are known to be able to bind to nucleotide sequences.

In some aspects, the binding domains can be locked nucleic acids (LNAs), Protein Nucleic Acids of all types (e.g. bisPNAs, gamma-PNAs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), or aptamers (e.g., DNA, RNA, protein, or combinations thereof).

In some aspects, the binding domains are one or more of DNA binding proteins (e.g., zinc finger proteins), antibody fragments (Fab), chemically synthesized binders (e.g., PNA, LNA, TALENS, or CRISPR), or a chemical modification (i.e., reactive moieties) in the synthetic polymer (e.g., thiolate, biotin, amines, carboxylates).

Target Molecule/Particles and Ligands

In the present technology, a target molecule or particle is detected or quantitated by virtue of its binding to a ligand in a fusion molecule that binds to a polymer scaffold. A target molecule or particle and a corresponding binding ligand can recognize and bind each other. For a particle, there can be surface molecules or markers suitable for a ligand to bind (therefore the marker and the ligand form a binding pair).

Examples of binding pairs that enable binding between a target molecule or a molecule on a particle include, but are not limited to, antigen/antibody (or antibody fragment); hormone, neurotransmitter, cytokine, growth factor or cell recognition molecule/receptor; and ion or element/chelate agent or ion binding protein, such as a calmodulin. The binding pairs can also be single-stranded nucleic acids having complementary sequences, enzymes and substrates, members of protein complex that bind each other, enzymes and cofactors, enzymes and one or more of their inhibitors (allosteric or otherwise), nucleic acid/protein, or cells or proteins detectable by cysteine-constrained peptides.

In some embodiments, the ligand is a protein, protein scaffold, peptide, aptamer (DNA or protein), nucleic acid (DNA or RNA), antibody fragment (Fab), chemically synthesized molecule, chemically reactive functional group or any other suitable structure that forms a binding pair with a target molecule.

Therefore, any target molecule in need of detection or quantitation, such as proteins, peptides, nucleic acids, chemical compounds, ions, and elements, can find a corresponding binding ligand. For the majority of proteins and nucleic acids, an antibody or a complementary sequence, or an aptamer can be readily prepared.

Likewise, binding ligands (such as antibodies and aptamers) can be readily found or prepared for particles, such as protein complexes and protein aggregates, protein/nucleic acid complexes, fragmented or fully assembled viruses, bacteria, cells, and cellular aggregates.

Fusion Molecule

A “fusion molecule” is intended to mean a molecule or complex that contains two functional regions, a binding domain and a ligand. The binding domain is capable of binding to a binding motif on a polymer scaffold, and the ligand is capable of binding to a target molecule.

In some aspects, the fusion molecule is prepared by linking the two regions with a bond or force. Such a bond and force can be, for instance, a covalent bond, a hydrogen bond, an ionic bond, a metallic bond, van der Walls force, hydrophobic interaction, or planar stacking interaction.

In some aspects, the fusion molecule, such as a fusion protein, can be expressed as a single molecule from a recombinant coding nucleotide. In some aspects, the fusion molecule is a natural molecule having a binding domain and a ligand suitable for use in the present technology.

Many options exist for connecting the binding domain with the ligand to form the fusion molecule. For example, the components may be connected via chemical coupling through functionalized linkers such as free amine, carboxylate coupling, thiolate, hydrazide, or azide (click) chemistry or the binding domain and the ligand may form one continuous transcript.

FIG. 2 illustrates a more specific embodiment of the system shown in FIG. 1. In FIG. 2, the fusion molecule is a chimeric protein that includes a zinc finger protein or domain 202 and a human immunodeficiency virus (HIV) envelop protein 203. The zinc finger protein 202 can bind to a suitable nucleotide sequence on the polymer scaffold, a double-stranded DNA 201; the HIV envelop protein 203 can bind to an anti-HIV antibody 204 which can be present in a biological sample (e.g., a blood sample from a patient) for detection.

When the double-stranded DNA 201 passes through a pore 205 of a nanopore device 206, the nanopore device 206 can detect whether a fusion molecule is bound to the DNA 201 and whether the bound fusion molecule binds to an anti-HIV antibody 204.

Measurement of Affinity of Binding

The present technology can be used also for measuring the binding affinity between two molecules and to determine other binding dynamics. For instance, after the binding motif passes through the pore of a nanopore device, the device can be reconfigured to reverse the moving direction of the polymer scaffold (as described below) such that the binding motif can pass through the pore again.

Before the binding motif enters the pore again, one can change the conditions in the sample that is loaded into the nanopore device. For instance, changing the condition can be one or more of removing the target molecule from the sample, adding an agent that competes with the target molecule or the ligand for binding, and changing the pH, salt, or temperature.

Under the changed conditions, the binding motif may be passed through the pore again. Therefore, whether the target molecule is still bound to the fusion molecule can be detected to determine how the changed conditions impact the binding.

In some aspects, once the binding motif is in the pore, it is retained there while the conditions are changed, and thus the impact of the changed conditions can be measured in situ.

Alternatively or in addition, the polymer scaffold can include multiple binding motifs and each of the binding motifs can bind to a fusion molecule that can bind to one or more specific a target molecule(s) or particle(s). While each binding motif passes through the pore, the conditions of the sample can be changed, allowing detection of changed binding between the ligand and the target molecule or particle on a continued basis.

Multiplexing

In some aspects, rather than including multiple binding motifs of the same kind as described above, a polymer scaffold can include multiple types of binding motifs, each having different corresponding binding domains. In such embodiments, a sample can include multiple types of fusion molecules, each type including one of the different corresponding binding domains and a ligand for a different target molecule or target microorganism.

An additional method of multiplexing includes assaying a collection of different scaffold molecules during a test, with each different scaffold associating with different fusion molecule(s). To determine what target molecules are in solution, scaffolds of the same type are labeled such that the sensor can identify what fusion molecule will bind to that particular scaffold. This can be accomplished, for example, by barcoding each type of scaffold with polyethylene glycol molecules of varying lengths or sizes.

With such a setting, a single polymer scaffold can be used to detect multiple types of target molecules or target microorganisms (e.g. bacterium or virus), or target cells (e.g. circulating tumor cells). FIG. 4 illustrates such a method. Here, a double-stranded DNA 403 is used as the polymer scaffold, the double-stranded DNA 403 including multiple binding motifs: two copies of a first binding motif 404, two copies of a second binding motif 405, and one copy of a third binding motif 406.

When the DNA passes through a nanopore device 407 that has two coaxial pores, 401 and 402, the binding status of each of the binding motifs is detected, in which both copies of binding motif 404 bind to a corresponding target molecule, both copies of binding motif 405 bind to a corresponding target molecule; and the fusion molecule bound to binding motif 406 does not bind to a corresponding target molecule.

This way, with a single polymer and a single nanopore device, the present technology can simultaneously detect multiple different target molecules or target microstructure (e.g., aggregates, microorganisms, or cells). Further, by determining how many copies of binding motifs are bound to the target molecules or target microorganisms, and by tuning conditions that impact the bindings, the system can obtain more detailed binding dynamic information.

Nanopore Devices

A nanopore device, as provided, includes at least a pore that forms an opening in a structure separating an interior space of the device into two volumes, and at least a sensor configured to identify objects (for example, by detecting changes in parameters indicative of objects) passing through the pore.

The pore(s) in the nanopore device are of a nano scale or micro scale. In one aspect, each pore has a size that allows a small or large molecule or microorganism to pass. In one aspect, each pore is at least about 1 nm in diameter. Alternatively, each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.

In one aspect, the pore is no more than about 100 nm in diameter. Alternatively, the pore is no more than about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.

In some aspects, each pore is at least about 100 nm, 200 nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm in diameter. In one aspect, the pore is no more than about 100000 nm in diameter. Alternatively, the pore is no more than about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.

In one aspect, the pore has a diameter that is between about 1 nm and about 100 nm, or alternatively between about 2 nm and about 80 nm, or between about 3 nm and about 70 nm, or between about 4 nm and about 60 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 40 nm, or between about 15 nm and about 30 nm.

In some aspects, the pore(s) in the nanopore device are of a larger scale for detecting large microorganisms or cells. In one aspect, each pore has a size that allows a large cell or microorganism to pass. In one aspect, each pore is at least about 100 nm in diameter. Alternatively, each pore is at least about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 1600 nm, 1700 nm, 1800 nm, 1900 nm, 2000 nm, 2500 nm, 3000 nm, 3500 nm, 4000 nm, 4500 nm, or 5000 nm in diameter.

In one aspect, the pore is no more than about 100,000 nm in diameter. Alternatively, the pore is no more than about 90,000 nm, 80,000 nm, 70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm, 10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.

In one aspect, the pore has a diameter that is between about 100 nm and about 10000 nm, or alternatively between about 200 nm and about 9000 nm, or between about 300 nm and about 8000 nm, or between about 400 nm and about 7000 nm, or between about 500 nm and about 6000 nm, or between about 1000 nm and about 5000 nm, or between about 1500 nm and about 3000 nm.

In some aspects, the nanopore device further includes means to move a polymer scaffold across the pore and/or means to identify objects that pass through the pore. Further details are provided below, described in the context of a two-pore device.

Compared to a single-pore nanopore device, a two-pore device can be more easily configured to provide good control of speed and direction of the movement of the polymer across the pores.

In one embodiment, the nanopore device includes a plurality of chambers, each chamber in communication with an adjacent chamber through at least one pore. Among these pores, two pores, namely a first pore and a second pore, are placed so as to allow at least a portion of a polymer to move out of the first pore and into the second pore. Further, the device includes a sensor capable of identifying the polymer during the movement. In one aspect, the identification entails identifying individual components of the polymer. In another aspect, the identification entails identifying fusion molecules and/or target molecules bound to the polymer. When a single sensor is employed, the single sensor may include two electrodes placed at both ends of a pore to measure an ionic current across the pore. In another embodiment, the single sensor comprises a component other than electrodes.

In one aspect, the device includes three chambers connected through two pores. Devices with more than three chambers can be readily designed to include one or more additional chambers on either side of a three-chamber device, or between any two of the three chambers. Likewise, more than two pores can be included in the device to connect the chambers.

In one aspect, there can be two or more pores between two adjacent chambers, to allow multiple polymers to move from one chamber to the next simultaneously. Such a multi-pore design can enhance throughput of polymer analysis in the device.

In some aspects, the device further includes means to move a polymer from one chamber to another. In one aspect, the movement results in loading the polymer across both the first pore and the second pore at the same time. In another aspect, the means further enables the movement of the polymer, through both pores, in the same direction.

For instance, in a three-chamber two-pore device (a “two-pore” device), each of the chambers can contain an electrode for connecting to a power supply so that a separate voltage can be applied across each of the pores between the chambers.

In accordance with one embodiment of the present disclosure, provided is a device comprising an upper chamber, a middle chamber and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore. Such a device may have any of the dimensions or other characteristics previously disclosed in U.S. Publ. No. 2013-0233709, entitled Dual-Pore Device, which is herein incorporated by reference in its entirety.

In some embodiments as shown in FIG. 5A, the device includes an upper chamber 505 (Chamber A), a middle chamber 504 (Chamber B), and a lower chamber 503 (Chamber C). The chambers are separated by two separating layers or membranes (501 and 502) each having a separate pore (511 or 512). Further, each chamber contains an electrode (521, 522 or 523) for connecting to a power supply. The annotation of upper, middle and lower chamber is in relative terms and does not indicate that, for instance, the upper chamber is placed above the middle or lower chamber relative to the ground, or vice versa.

Each of the pores 511 and 512 independently has a size that allows a small or large molecule or microorganism to pass. In one aspect, each pore is at least about 1 nm in diameter. Alternatively, each pore is at least about 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, or 100 nm in diameter.

In one aspect, the pore is no more than about 100 nm in diameter. Alternatively, the pore is no more than about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm in diameter.

In one aspect, the pore has a diameter that is between about 1 nm and about 100 nm, or alternatively between about 2 nm and about 80 nm, or between about 3 nm and about 70 nm, or between about 4 nm and about 60 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 40 nm, or between about 15 nm and about 30 nm.

In other aspects, each pore is at least about 100 nm, 200 nm, 500 nm, 1000 nm, 2000 nm, 3000 nm, 5000 nm, 10000 nm, 20000 nm, or 30000 nm in diameter. In one aspect, each pore is 50,000 nm to 100,000 nm in diameter. In one aspect, the pore is no more than about 100000 nm in diameter. Alternatively, the pore is no more than about 50000 nm, 40000 nm, 30000 nm, 20000 nm, 10000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm in diameter.

In some aspects, the pore has a substantially round shape. “Substantially round”, as used here, refers to a shape that is at least about 80 or 90% in the form of a cylinder. In some embodiments, the pore is square, rectangular, triangular, oval, or hexangular in shape.

Each of the pores 511 and 512 independently has a depth (i.e., a length of the pore extending between two adjacent volumes). In one aspect, each pore has a depth that is least about 0.3 nm. Alternatively, each pore has a depth that is at least about 0.6 nm, 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm.

In one aspect, each pore has a depth that is no more than about 100 nm. Alternatively, the depth is no more than about 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, or 10 nm.

In one aspect, the pore has a depth that is between about 1 nm and about 100 nm, or alternatively, between about 2 nm and about 80 nm, or between about 3 nm and about 70 nm, or between about 4 nm and about 60 nm, or between about 5 nm and about 50 nm, or between about 10 nm and about 40 nm, or between about 15 nm and about 30 nm.

In some aspects, the nanopore extends through a membrane. For example, the pore may be a protein channel inserted in a lipid bilayer membrane or it may be engineered by drilling, etching, or otherwise forming the pore through a solid-state substrate such as silicon dioxide, silicon nitride, grapheme, or layers formed of combinations of these or other materials. In some aspects, the length or depth of the nanopore is sufficiently large so as to form a channel connecting two otherwise separate volumes. In some such aspects, the depth of each pore is greater than 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm. In some aspects, the depth of each pore is no more than 2000 nm or 1000 nm.

In one aspect, the pores are spaced apart at a distance that is between about 10 nm and about 1000 nm. In some aspects, the distance between the pores is greater than 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, 6000 nm, 7000 nm, 8000 nm, or 9000 nm. In some aspects, the pores are spaced no more than 30000 nm, 20000 nm, or 10000 nm apart. In one aspect, the distance is at least about 10 nm, or alternatively, at least about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, or 300 nm. In another aspect, the distance is no more than about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nm, 150 nm, or 100 nm.

In yet another aspect, the distance between the pores is between about 20 nm and about 800 nm, between about 30 nm and about 700 nm, between about 40 nm and about 500 nm, or between about 50 nm and about 300 nm.

The two pores can be arranged in any position so long as they allow fluid communication between the chambers and have the prescribed size and distance between them. In one aspect, the pores are placed so that there is no direct blockage between them. Still, in one aspect, the pores are substantially coaxial, as illustrated in FIG. 5A.

In one aspect, as shown in FIG. 5A, the device, through the electrodes 521, 522, and 523 in the chambers 503, 504, and 505, respectively, is connected to one or more power supplies. In some aspects, the power supply includes a voltage-clamp or a patch-clamp, which can supply a voltage across each pore and measure the current through each pore independently. In this respect, the power supply and the electrode configuration can set the middle chamber to a common ground for both power supplies. In one aspect, the power supply or supplies are configured to apply a first voltage V₁ between the upper chamber 505 (Chamber A) and the middle chamber 504 (Chamber B), and a second voltage V₂ between the middle chamber 504 and the lower chamber 503 (Chamber C).

In some aspects, the first voltage V₁ and the second voltage V₂ are independently adjustable. In one aspect, the middle chamber is adjusted to be a ground relative to the two voltages. In one aspect, the middle chamber comprises a medium for providing conductance between each of the pores and the electrode in the middle chamber. In one aspect, the middle chamber includes a medium for providing a resistance between each of the pores and the electrode in the middle chamber. Keeping such a resistance sufficiently small relative to the nanopore resistances is useful for decoupling the two voltages and currents across the pores, which is helpful for the independent adjustment of the voltages.

Adjustment of the voltages can be used to control the movement of charged particles in the chambers. For instance, when both voltages are set in the same polarity, a properly charged particle can be moved from the upper chamber to the middle chamber and to the lower chamber, or the other way around, sequentially. In some aspects, when the two voltages are set to opposite polarity, a charged particle can be moved from either the upper or the lower chamber to the middle chamber and kept there.

The adjustment of the voltages in the device can be particularly useful for controlling the movement of a large molecule, such as a charged polymer, that is long enough to cross both pores at the same time. In such an aspect, the direction and the speed of the movement of the molecule can be controlled by the relative magnitude and polarity of the voltages as described below.

The device can contain materials suitable for holding liquid samples, in particular, biological samples, and/or materials suitable for nanofabrication. In one aspect, such materials include dielectric materials such as, but not limited to, silicon, silicon nitride, silicon dioxide, graphene, carbon nanotubes, TiO₂, HfO₂, Al₂O₃, or other metallic layers, or any combination of these materials. In some aspects, for example, a single sheet of graphene membrane of about 0.3 nm thick can be used as the pore-bearing membrane.

Devices that are microfluidic and that house two-pore microfluidic chip implementations can be made by a variety of means and methods. For a microfluidic chip comprised of two parallel membranes, both membranes can be simultaneously drilled by a single beam to form two concentric pores, though using different beams on each side of the membranes is also possible in concert with any suitable alignment technique. In general terms, the housing ensures sealed separation of Chambers A-C. In one aspect as shown in FIG. 5B, the housing would provide minimal access resistance between the voltage electrodes 521, 522, and 523 and the nanopores 511 and 512, to ensure that each voltage is applied principally across each pore.

In one aspect, the device includes a microfluidic chip (labeled as “Dual-core chip”) is comprised of two parallel membranes connected by spacers. Each membrane contains a pore drilled by a single beam through the center of the membrane. Further, the device preferably has a Teflon® housing for the chip. The housing ensures sealed separation of Chambers A-C and provides minimal access resistance for the electrode to ensure that each voltage is applied principally across each pore.

More specifically, the pore-bearing membranes can be made with transmission electron microscopy (TEM) grids with a 5-100 nm thick silicon, silicon nitride, or silicon dioxide windows. Spacers can be used to separate the membranes, using an insulator, such as SU-8, photoresist, PECVD oxide, ALD oxide, ALD alumina, or an evaporated metal material, such as Ag, Au, or Pt, and occupying a small volume within the otherwise aqueous portion of Chamber B between the membranes. A holder is seated in an aqueous bath that is comprised of the largest volumetric fraction of Chamber B. Chambers A and C are accessible by larger diameter channels (for low access resistance) that lead to the membrane seals.

A focused electron or ion beam can be used to drill pores through the membranes, naturally aligning them. The pores can also be sculpted (shrunk) to smaller sizes by applying a correct beam focusing to each layer. Any single nanopore drilling method can also be used to drill the pair of pores in the two membranes, with consideration to the drill depth possible for a given method and the thickness of the membranes. Predrilling a micro-pore to a prescribed depth and then a nanopore through the remainder of the membranes is also possible to further refine the membrane thickness.

In another aspect, the insertion of biological nanopores into solid-state nanopores to form a hybrid pore can be used in either or both pores in the two-pore method. The biological pore can increase the sensitivity of the ionic current measurements, and is useful when only single-stranded polynucleotides are to be captured and controlled in the two-pore device, e.g., for sequencing.

By virtue of the voltages present at the pores of the device, charged molecules can be moved through the pores between chambers. Speed and direction of the movement can be controlled by the magnitude and polarity of the voltages. Further, because each of the two voltages can be independently adjusted, the direction and speed of the movement of a charged molecule can be finely controlled in each chamber.

One example concerns a charged polymer scaffold, such as a DNA, having a length that is longer than the combined distance that includes the depth of both pores plus the distance between the two pores. For example, a 1000 by dsDNA is about 340 nm in length, and would be substantially longer than the 40 nm spanned by two 10 nm-deep pores separated by 20 nm. In a first step, the polynucleotide is loaded into either the upper or the lower chamber. By virtue of its negative charge under a physiological condition at a pH of about 7.4, the polynucleotide can be moved across a pore on which a voltage is applied. Therefore, in a second step, two voltages, in the same polarity and at the same or similar magnitudes, are applied to the pores to move the polynucleotide across both pores sequentially.

At about the time when the polynucleotide reaches the second pore, one or both of the voltages can be changed. Since the distance between the two pores is selected to be shorter than the length of the polynucleotide, when the polynucleotide reaches the second pore, it is also in the first pore. A prompt change of polarity of the voltage at the first pore, therefore, will generate a force that pulls the polynucleotide away from the second pore as illustrated in FIG. 5C.

Assuming that the two pores have identical voltage-force influence and |V₁|=|V₂|+δV, the value δV>0 (or <0) can be adjusted for tunable motion in the V₁| (or V₂) direction. In practice, although the voltage-induced force at each pore will not be identical with V₁=V₂, calibration experiments can identify the appropriate bias voltage that will result in equal pulling forces for a given two-pore chip; and variations around that bias voltage can then be used for directional control.

If, at this point, the magnitude of the voltage-induced force at the first pore is less than that of the voltage-induced force at the second pore, then the polynucleotide will continue crossing both pores towards the second pore, but at a lower speed. In this respect, it is readily appreciated that the speed and direction of the movement of the polynucleotide can be controlled by the polarities and magnitudes of both voltages. As will be further described below, such a fine control of movement has broad applications.

Accordingly, in one aspect, provided is a method for controlling the movement of a charged polymer through a nanopore device. The method entails (a) loading a sample comprising a charged polymer in one of the upper chamber, middle chamber or lower chamber of the device of any of the above embodiments, wherein the device is connected to one or more power supplies for providing a first voltage between the upper chamber and the middle chamber, and a second voltage between the middle chamber and the lower chamber; (b) setting an initial first voltage and an initial second voltage so that the polymer moves between the chambers, thereby locating the polymer across both the first and second pores; and (c) adjusting the first voltage and the second voltage so that both voltages generate force to pull the charged polymer away from the middle chamber (voltage-competition mode), wherein the two voltages are different in magnitude, under controlled conditions, so that the charged polymer moves across both pores in either direction and in a controlled manner.

To establish the voltage-competition mode in step (c), the relative force exerted by each voltage at each pore is to be determined for each two-pore device used, and this can be done with calibration experiments by observing the influence of different voltage values on the motion of the polynucleotide, which can be measured by sensing known-location and detectable features in the polynucleotide, with examples of such features detailed later in this disclosure. If the forces are equivalent at each common voltage, for example, then using the same voltage value at each pore (with common polarity in upper and lower chambers relative to grounded middle chamber) creates a zero net motion in the absence of thermal agitation (the presence and influence of Brownian motion is discussed below). If the forces are not equivalent at each common voltage, achieving equal forces involves the identification and use of a larger voltage at the pore that experiences a weaker force at the common voltage. Calibration for voltage-competition mode can be done for each two-pore device, and for specific charged polymers or molecules whose features influence the force when passing through each pore.

In one aspect, the sample containing the charged polymer is loaded into the upper chamber and the initial first voltage is set to pull the charged polymer from the upper chamber to the middle chamber and the initial second voltage is set to pull the polymer from the middle chamber to the lower chamber. Likewise, the sample can be initially loaded into the lower chamber, and the charged polymer can be pulled to the middle and the upper chambers.

In another aspect, the sample containing the charged polymer is loaded into the middle chamber; the initial first voltage is set to pull the charged polymer from the middle chamber to the upper chamber; and the initial second voltage is set to pull the charged polymer from the middle chamber to the lower chamber.

In one aspect, the adjusted first voltage and second voltage at step (c) are about 10 times to about 10,000 times as high, in magnitude, as the difference/differential between the two voltages. For instance, the two voltages can be 90 mV and 100 mV, respectively. The magnitude of the two voltages, about 100 mV, is about 10 times of the difference/differential between them, 10 mV. In some aspects, the magnitude of the voltages is at least about 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, 100 times, 150 times, 200 times, 250 times, 300 times, 400 times, 500 times, 1000 times, 2000 times, 3000 times, 4000 times, 5000 times, 6000 times, 7000 times, 8000 times or 9000 times as high as the difference/differential between them. In some aspects, the magnitude of the voltages is no more than about 10000 times, 9000 times, 8000 times, 7000 times, 6000 times, 5000 times, 4000 times, 3000 times, 2000 times, 1000 times, 500 times, 400 times, 300 times, 200 times, or 100 times as high as the difference/differential between them.

In one aspect, real-time or on-line adjustments to the first voltage and the second voltage at step (c) are performed by active control or feedback control using dedicated hardware and software, at clock rates up to hundreds of megahertz. Automated control of the first or second or both voltages is based on feedback of the first or second or both ionic current measurements.

Sensors

As discussed above, in various aspects, the nanopore device further includes one or more sensors to carry out the identification of the binding status of the binding motifs.

The sensors used in the device can be any sensor suitable for identifying a molecule or particle, such as a polymer. For instance, a sensor can be configured to identify the polymer by measuring a current, a voltage, a pH value, an optical feature, or residence time associated with the polymer. In other aspects, the sensor may be configured to identify one or more individual components of the polymer or one or more components bound to the polymer. The sensor may be formed of any component configured to detect a change in a measurable parameter where the change is indicative of the polymer, a component of the polymer, or preferably, a component bound to the polymer. In one aspect, the sensor includes a pair of electrodes placed at two sides of a pore to measure an ionic current across the pore when a molecule or particle, in particular a polymer, moves through the pore. In certain aspects, the ionic current across the pore changes measurably when a polymer segment passing through the pore is bound to a fusion molecule and/or fusion molecule-target molecule complex. Such changes in current may vary in predictable, measurable ways corresponding with, for example, the presence, absence, and/or size of the fusion molecules and target molecules present.

In one embodiment, the sensor measures an optical feature of the polymer, a component (or unit) of the polymer, or a component bound to the polymer. One example of such measurement includes the identification of an absorption band unique to a particular unit by infrared (or ultraviolet) spectroscopy.

When residence time measurements are used, the size of the component can be correlated to the specific component based on the length of time it takes to pass through the sensing device.

Still further, in embodiments directed towards detecting units of the polymer, the sensor can include an enzyme distal to the sensing device, where the enzyme is capable of separating the terminal unit of the polymer from the penultimate unit, thereby providing for a single molecular unit of the polymer. The single molecule, such as a single nucleotide or an amino acid, can then translocate through the pore and may or may not be detected. However, when the enzyme encounters a bound target molecule, the enzyme will not be able to cleave the penultimate unit, and therefore will become stalled or will skip to the next available cleavage sites, thus releasing a fragment that has a comparable size difference from a single unit and is thus detectable. Detection can be done with sensors as described in this application or detected with methods such as mass spectrometry. Methods for measuring such units are known in the art and include those developed by Cal Tech (see, e.g., spectrum.ieee.org/tech-tallVat-work/test-and-measurement/a-scale-for-weighing-single-molecules). The results of such analysis can be compared to those of the sensing device to confirm the correctness of the analysis.

In some embodiments, the sensor is functionalized with reagents that form distinct non-covalent bonds with each association site or each associated target molecule. In this respect, the gap is large enough to allow effective measuring. For instance, when a sensor is functionalized with reagents to detect a feature on DNA that is 5 nm on a dsDNA scaffold, a 7.5 nm gap can be used, because DNA is 2.5 nm wide.

Tunnel sensing with a functionalized sensor is termed “recognition tunneling.” Using current technology, a Scanning Tunneling Microscope (STM) with recognition tunneling identifies a DNA base flanked by other bases in a short DNA oligomer. As has been described, recognition tunneling can provide a “universal reader” designed to hydrogen-bond in a unique orientation to molecules that a user desires to be detected. Most reported is the identification of nucleic acids; however, it is herein modified to be employed to detect target molecules on a scaffold.

A limitation with the conventional recognition tunneling is that it can detect only freely diffusing molecules that randomly bind in the gap, or that happen to be in the gap during microscope motion, with no method of explicit capture in the gap. However, the collective drawbacks of the STM setup can be eliminated by incorporating the recognition reagent, optimized for sensitivity, within an electrode tunneling gap in a nanopore channel.

Accordingly, in one embodiment, the sensor includes surface modification by a reagent. In one aspect, the reagent is capable of forming a non-covalent bond with an association site or an attached target molecule. In a particular aspect, the bond is a hydrogen bond. Non-limiting examples of the reagent include 4-mercaptobenzamide and 1-H-Imidazole-2-carboxamide.

Furthermore, the methods of the present technology can provide DNA delivery rate control for one or more recognition tunneling sites, each positioned in one or both of the nanopore channels, and voltage control can ensure that each target molecule resides in each site for a sufficient duration for robust identification.

Sensors in the devices and methods of the present disclosure can comprise gold, platinum, graphene, or carbon, or other suitable materials. In a particular aspect, the sensor includes parts made of graphene. Graphene can act as a conductor and an insulator, thus tunneling currents through the graphene and across the nanopore can sequence the translocating DNA.

In some embodiments, the tunnel gap has a width from about 1 nm to about 20 nm. In one aspect, the width of the gap is at least about 1 nm, or alternatively, at least about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 12, or 15 nm. In another aspect, the width of the gap is not greater than about 20 nm, or alternatively, not greater than about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nm. In some aspects, the width is between about 1 nm and about 15 nm, between about 1 nm and about 10 nm, between about 2 nm and about 10 nm, between about 2.5 nm and about 10 nm, or between about 2.5 nm and about 5 nm.

In other embodiments, the tunnel gap is suitable for detecting micro-sized particles (e.g., viruses, bacteria, and/or cells) and has a width from about 1000 nm to about 100,000 nm. In some embodiments, the width of the gap is between about 10,000 nm and 80,000 nm or between about 20,000 nm and 50,000 nm. In another embodiment, the width of the gap is between about 50,000 nm and 100,000 nm. In some embodiments, the width of the gap is not greater than about 100,000 nm, 90,000 nm, 80,000 nm, 70,000 nm, 60,000 nm, 50,000 nm, 40,000 nm, 30,000 nm, 20,000 nm, 10,000 nm, 9000 nm, 8000 nm, 7000 nm, 6000 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, or 1000 nm.

In some embodiments, the sensor is an electric sensor. In some embodiments, the sensor detects a fluorescent detection means when the target molecule or the detectable label passing through has a unique fluorescent signature. A radiation source at the outlet of the pore can be used to detect that signature.

EXAMPLES

The present technology is further defined by reference to the following example and experiments. It will be apparent to those skilled in the art that many modifications may be practiced without departing from the scope of the current invention

The example section begins by first pointing out principal reasons to use a polymer scaffold and fusion molecules in biomarker detection. A primary reason is that a biomarker alone, below a certain size threshold, is undetectable with a nanopore, as shown for proteins of varying sizes in Calin Plesa, Stefan W. Kowalczyk, Ruben Zinsmeester, Alexander Y. Grosberg, Yitzhak Rabin, and Cees Dekker. “Fast translocation of proteins through solid state nanopores.” Nano letters 13, no. 2 (2013): 658-663. Moreover, even those biomarkers that are detectable may not be distinguishable. A biomarker will yield the same nanopore signature as all other molecules of comparable size/charge, preventing discrimination. By using a scaffold and fusion molecules, we can avoid both of these problems. In particular, we show by the examples that detection of representative fusion molecules on scaffolds can be demonstrated, and further that detection of target molecules to fusion molecules on the scaffold can also be detected. With this capability, discrimination can be achieved by appropriate engineering of the ligand domain of the fusion molecule, to achieve specificity for the target molecule of interest.

Example 1 DNA Alone in Solid-State Nanopore Experiment

Nanopore instruments use a sensitive voltage-clamp amplifier to apply a voltage V across the pore while measuring the ionic current l₀ through the open pore (FIG. 6A). When a single charged molecule such as a double-stranded DNA (dsDNA) is captured and driven through the pore by electrophoresis (FIG. 6B), the measured current shifts from l₀ to l_(B), and the shift amount Δl=l₀−l_(B) and duration t_(D) are used to characterize the event. After recording many events during an experiment, distributions of the events (FIG. 6C) are analyzed to characterize the corresponding molecule. In this way, nanopores provide a simple, label-free, purely electrical single-molecule method for biomolecular sensing.

In the DNA experiment shown in FIG. 6A, FIG. 6B, and FIG. 6C, the single nanopore fabricated in silicon nitride (SiN) substrate is a 40 nm diameter pore in 100 nm thick SiN membrane (FIG. 6A). In FIG. 6B, the representative current trace shows a blockade event caused by a 5.6 kb dsDNA passing in a single file manner (unfolded) through an 11 nm diameter nanopore in 10 nm thick SiN at 200 mV and 1M KCl. The mean open channel current is l₀=9.6 nA, with mean event amplitude l_(B)=9.1 nA, and duration t_(D)=0.064 ms. The amplitude shift is Δl=l₀−l_(B)=0.5 nA. In FIG. 6C, the scatter plot shows lΔ/l vs. t_(D) for all 1301 events recorded over 16 minutes.

In the DNA experiment shown in FIG. 7A, dsDNA alone causes current enhancement events at 100 mM KCl. This was shown in the published research of Smeets, Ralph M M, et al. “Salt dependence of ion transport and DNA translocation through solid-state nanopores.” Nano Letters 6.1 (2006): 89-95). The study showed that, while the amplitude shift Δl=l₀−l_(B)>0 for KCl concentration above 0.4 M, the shift has opposite polarity (Δl<0) for KCl concentration below 0.4 M. As this is a negative voltage experiment (−200 mV) with KCl concentration below 0.4 M, we see that the DNA event has the same polarity (316) relative to the baseline (315) as shown in FIG. 3C.

Example 2 VspR Protein Binding to DNA Scaffold and Nanopore Detection

The VspR protein is a 90 kDa protein from V. cholerae that binds directly to dsDNA with high micromolar affinity (see reference: Yildiz, Fitnat H., Nadia A. Dolganov, and Gary K. Schoolnik. “VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of Biosynthesis Genes and EPSETr-Associated Phenotypes in Vibrio cholerae 01 El Tor.” Journal of bacteriology 183, no. 5 (2001): 1716-1726). In this example of target detection using nanopore technology, VspR acts as the fusion molecule with a site-specific DNA binding domain, and a ligand specific binding site that can be engineered for the purpose of detecting a variety of targets, including antibodies or sugars. In this demonstration, we show detection of the VspR on the DNA scaffold as a model fusion molecule. The scaffold contains 10 VspR specific binding sites (FIG. 8). To preserve affinity of VspR for dsDNA binding, we use 0.1 M KCl, a salt concentration in which DNA alone translocations cause current enhancements, as shown in Example 1 and FIG. 7A. The 5.631 kb DNA scaffold contains 10 total VspR binding sites: 5 of one sequence (14 base pairs), 3 of a different sequence (18 base pairs), and 2 of a third sequence (27 bp). The three different sequences may not bind VspR with equal affinity. In experiments, VspR protein concentration is 18 nM in the recording buffer, and 180 nM during labeling (binding step). This results in 18× excess of VspR protein to binding sites on DNA. The experiment was run at pH 8.0 (pl of VspR protein is 5.8). Taking Kd and DNA concentration into account, only 0.1-1% of DNA should be fully occupied by VspR, with a larger percentage partially occupied, and some unknown remaining percentage of DNA entirely unbound. There is also free VspR protein in solution during the nanopore experiment.

Two representative events are shown in FIG. 9A and FIG. 9B. In the experiments with VspR, VspR concentration was 18 nM (1.6 mg/L), 10 nM binding sites. The scaffold concentration was 1 nM resulting in capture every 6.6 seconds. From this, the theoretical sensitivity using a 10 uI sample is 116 pM (0.01 mg/ml). The pore size is 15 nm in diameter and length. The voltage is −100 mV, and note that negative voltages create negative currents, so upward shifts correspond to attenuation events, as shown for the VspR-bound DNA event (FIG. 9B), whereas downward shifts create positive shifts as shown for the unbound DNA scaffold event (FIG. 9A). This is consistent with the idealized signal patterns and conditions in FIG. 3C, with the DNA event (316) having faster duration and the opposite polarity compared to the fusion molecule-bound DNA event (320). Thus, the key observation from this figure is that VspR-bound events have the opposite signal polarity compared to unbound DNA events. FIG. 10 shows ten more representative current attenuation events consistent with the VspR-bound scaffold passing through the pore. There were 90 such events over 10 minutes of recording, corresponding to 1 VspR-bound event every 6.6 seconds. Events were attenuations of 50 to 150 pA in amplitude and 0.2 to 2 milliseconds in duration. As stated, downward events correspond to current enhancement events and upward events correspond to current attenuation events in FIG. 9A, FIG. 9B, and FIG. 10, and this shift direction is preserved even though the baseline is zeroed for display purposes.

Example 3 RecA Protein Binding to DNA Scaffold and Nanopore Detection

RecA comprises the elements of a fusion molecule, and this example demonstrates the ability to use these elements to detect a target biomarker. Specifically, the fusion molecule consists of the portion of RecA that binds DNA (i.e. the DNA binding domain) and the portion of RecA (epitope) that baits the biomarker (anti-RecA antibody). DNA and RecA experiments were performed first in the absence and then in the presence of anti-RecA antibody.

Reagent DNA/RecA consists of the 5.6 kb dsDNA scaffold molecule coated in RecA. RecA is a 38 kDa bacterial protein involved in DNA repair, which is capable of polymerizing along dsDNA (see [C Bell. Structure and mechanism of Escherichia coli RecA ATPase. Molecular microbiology, 58(2):358-366, January 2005]). This reagent is created by incubating 60 nM scaffold with 112 uM RecA protein in 10 mM gamma-S-ATP, 70 mM Tris pH 7.6, 10 mM MgCl, and 5 mM DTT (New England Biolabs). Gamma-S-ATP is included since RecA binds to dsDNA with greater affinity if the RecA has ATP bound. Since RecA can hydrolyze ATP to ADP, thereby reducing its affinity for DNA, the non-hydrolyzable gamma-S ATP analog prevents this transition to ADP and thus the higher affinity state is maintained. Even though the ratio of RecA to DNA is one RecA molecule for every possible 3-bp binding site, we expect that not all the RecA protein is binding and thus there is free RecA in solution, as observed in other nanopore studies (see Smeets, R. M. M., S. W. Kowalczyk, A. R. Hall, N. H. Dekker, and C. Dekker. “Translocation of RecA coated double-stranded DNA through solid-state nanopores.” Nano letters 9, no. 9 (2008): 3089-3095, and Kowalczyk, Stefan W., Adam R. Hall, and Cees Dekker. “Detection of local protein structures along DNA using solid-state nanopores.” Nano letters 10, no. 1 (2009): 324-328). DNA/RecA samples are then adjusted to 1M KCl or LiCl, 10 mM EDTA and tested in a nanopore experiment or excess RecA protein is removed using gel filtration (ThermoScientific Spin Columns).

In one set of experiments, we used a 16-18 nm diameter pore formed in a 30 nm thick SiN membrane, applying 180 mV in 1M KCl at pH 8. In separate control experiments, unbound 5.6 kb dsDNA scaffold generates 95% of events in the range of 2100-400 pA and 530-500 microseconds. Also, free RecA events are 2100-600 pA, 20-200 usec. Finally, RecA-bound DNA events are typically much deeper blockades, in the range 0.51-3 nA, and with longer duration (0.200-3 milliseconds). Representative events for RecA-bound DNA are shown in FIG. 11 and FIG. 12. These events have interesting patterns, which in the paper by Kowalczyk et al. [“Detection of local protein structures along DNA using solid-state nanopores.” Nano letters 10, no. 1 (2009): 324-328)] the authors attempt to infer the location and length of RecA filaments that are bound to each DNA; however, this is speculative, since it assumes a uniform passage rate through the pore even though another study showed that dsDNA does not pass through a pore at a uniform rate [Lu, Bo, et al. “Origins and consequences of velocity fluctuations during DNA passage through a nanopore.” Biophysical journal 101.1 (2011): 70-79]. FIG. 13 part (a) and (c) show on the vertical axis the maximum and mean current shift, respectively, normalized by voltage, and the event duration on the horizontal axis. Both event plots have all 1385 events recorded over 10 minutes. The amplitude is normalized by voltage to give event conductance shift values, which is common in nanopore research papers. For example, a mean conductance of 14 nS at 200 mV is equivalent to a mean current amplitude of 2.8 nA. There are two observable sub-populations in amplitude (or equivalently, conductance) and duration, with the deeper and longer duration events attributable to RecA-bound DNA and the faster shallower events attributable to free RecA in solution. We verified the identity of the faster, shallower subpopulation as free RecA by running RecA alone control experiments. This was also verified in the earlier study [Smeets, R. M. M., S. W. Kowalczyk, A. R. Hall, N. H. Dekker, and C. Dekker. “Translocation of RecA coated double-stranded DNA through solid-state nanopores.” Nano letters 9, no. 9 (2008): 3089-3095]. Looking at the maximum current shift value (FIG. 13, parts (a) and (b)) instead of the mean (FIG. 13, parts (c) and (d)) makes the subpopulations events more distinct. Note that RecA-bound DNA vs. unbound DNA event patterns are consistent with the model signal patterns in FIG. 3A.

In separate experiments, to demonstrate detection of a target antibody, RecA antibody was used. The DNA/RecA reagent binds an antibody biomarker creating a DNA/RecA/Ab complex by incubating one nanomolar DNA/RecA for 30 mins with either an anti-RecA monoclonal antibody (ARM191, Fisher Scientific) or polyclonal RecA anti-serum (gift from Prof. Ken Knight, Ph. D., UMass Medical School), at a 1:10000 dilution. Electrophoretic mobility shift assays, 5% TBE polyacrylamide gel in 1×TBE buffer, are used to test the DNA/RecA and DNA/RecA/Ab complexes by comparing migration of complexes to DNA only or the proper controls.

The nanopore experiments were run at 200 mV in 1M LiCl with a pore that varied in diameter: 20 nm during the DNA alone control, and then enlarged to 27 nm after RecA-bound DNA complexes were added. In a gel shift experiment, FIG. 14A shows a clear shift for DNA/RecA/mAb above DNA/RecA, which is in turn well above the unbound 5.6 kb dsDNA scaffold. This complex was tested experimentally with a nanopore. Specifically, 0.1 nM DNA was added to the chamber above the pore, and after 10 minutes of recording, 1.25 nM DNA/RecA was added. After another period of recording, 1.25 nM DNA/RecA/mAb was added. With the AB-bound complexes in solution, a new multi-level event type was observed (FIG. 14B) that did not match event patterns characteristic of the other two complex types (DNA, DNA/RecA). The Δl vs. t_(D) distributions of events recorded during each phase of the experiment (FIG. 14C) show that RecA-bound DNA events have longer durations t_(D), and 3 times as many events had a mean amplitude shift Δl greater than 0.6 nA after DNA/RecA/mAb was added. A simple criteria for tagging events in this data set as also being Ab-bound is (Δl, t_(D)) (0.6 nA, 0.2 ms). Identifying a best signature that is almost absent in unbound DNA events, but is present in a significant fraction of RecA-bound events (with or without antibody also bound to DNA/RecA), is useful for detection of the presence of RecA-bound DNA complexes in solution above the nanopore. For the purpose of antibody detection, we take this a step further, and aim to identify a best signature that is almost absent in unbound DNA and RecA-bound DNA event types, but is present in a significant fraction of RecA-bound events with antibody also bound to DNA/RecA. This provides a criterion for detection of the presence of RecA-bound DNA complexes in solution above the nanopore. As these DNA and RecA and RecA-antibody experiments are done with a positive voltage with KCl concentration above 0.4 M, we see that the event patterns in FIG. 14B are comparable to the idealized patterns in FIG. 3A.

Example 4 Fusion Molecules Comprising PNA and Biotin for Target Protein Detection

The previous example explores detection of RecA-antibody bound to RecA-coated dsDNA complexes. Since RecA binds 3 by regions non-specifically, and thus RecA-antibody could bind to any RecA on dsDNA, it is also desirable to demonstrate an approach that permits target binding to specific sites. In particular, we use a 5.6 kb dsDNA scaffold that is engineered to bind 12-mer peptide-nucleic-acid (PNA) molecules, with each PNA having 3 biotinylated sites for binding an avidin family member (e.g., neutravidin, and or monovalent streptavidin) (FIG. 15A). The scaffold has 25 distinct sites that together localize up to 75 avidin biomarker binding sites (FIG. 15B). Our data (FIG. 16D) shows that the DNA/PNA/Neut complexes cause event signatures that are detectable above other background event types (unbound DNA alone, Neutravidin alone, PNA/Neutravidin alone) and can therefore be tagged as fully assembled (i.e. DNA/PNA/Neutravidin) events. In this setup, it is the fully assembled DNA/PNA/Neutravidin complex that acts as the scaffold+fusion molecule. In the remainder of the example, we provide sufficient detail to show that DNA/PNA/Neutravidin complexes can be detected with a nanopore.

In this setup, the fusion molecule contains two separate domains, one that binds a unique DNA sequence and another that binds to an anti-Neutravidin antibody. The DNA binding domain is a protein nucleic acid molecule (PNA) that binds to the unique sequence (GAAAGTGAAAGT, uSeq1) that is repeated 25 times throughout the scaffold (FIG. 15B). PNA molecules are similar to oligonucleotides having A, T, C, G bases, which are capable of pairing with their complementary sequences, but instead of a phosphate backbone like typical oligonucleotides, the backbone is protein. This eliminates the negative charge provided by the phosphate backbone, and thus, PNA molecules can incorporate into dsDNA by displacing the complementary DNA strand, making a new DNA/PNA hybrid for the short stretch that encompasses the PNA molecule. The PNA used in the experiment had the sequence GAA*AGT*GAA*AGT where the * indicates that a biotin was incorporated into the PNA backbone at the gamma position by coupling to a Lysine amino acid, and thus, each PNA has three biotin molecules (PNABio). To create the fusion molecule bound scaffold, a 60 nM scaffold is heated to 95 C for 2 minutes, cooled to 60 C and incubated with a 10× excess of PNA to possible binding sites in 15 mM NaCl for 1 hr and then cooled to 4 C. The excess PNA is dialyzed out (20k MWCO, Thermo Scientific) for 2 hrs against 10 mM Tris pH 8.0. This DNA/PNA complex is then labeled with a 10 fold excess Neutravidin protein (Pierce/Thermo Scientific) to possible biotin sites (assuming a 60% reduction of PNA during dialysis). The reaction is electrophoresed as described above to assess purity, concentration, and potential aggregation. This reagent, DNA/PNA/Neutravidin (D/P/N), is stored at −20 C until use.

FIG. 17A and FIG. 17B show data comparing Δl vs. tD distributions from three separate experiments: DNA alone, Neutravidin alone, and D/P/N reagents. The largest 1All events in the D/P/N experiment are most likely attributed to D/P/N complexes (FIG. 16D), providing a simple criteria for tagging events as fusion molecule bound (i.e., scaffold with PNA and Neutravidin bound). Specifically, we can flag an event as being “fusion-molecule bound” if |Δ^(l)|>4 nA for that event. For the data sets in FIG. 17A, 9.3% (390) of events in the D/P/N experiment have |Δ^(l)|>4 nA, with only 0.46% of D and 0.16% of N events in controls exceeding 4 nA. In a separate experiment (data not shown) with a 7 nm diameter pore at 1M KCl and 200 mV applied, in a control with only PNA and Neutravidin at 0.4 nM concentration, no events (0%) exceeded 4 nA. Applying our mathematical criteria, the random variable Q={Fraction of flagged events} has a binomial distribution, and using this and other statistical modeling tools, we can compute the 99% confidence interval for this data set as Q=9.29±1.15%. Since 9.29%>0.46% (the max false-positive %) is satisfied well within the 99% confidence interval for Q, we have a positive test result, and in under 8 minutes of data gathering. In fact the same 99% confidence is achieved for this data set with only the first 60 seconds of the data. The gel shift (FIG. 17C) shows that scaffold DNA migration is retarded in a Neutravidin dependent manner; this guided us to using the 10× concentration in this preliminary experiment, as it appeared all DNA is labeled and a nearly homogenous population is created. We do not see a shift with the DNA/PNA complex, though a shift was observed in another DNA/PNA nanopore study using shorter DNA (Alon Singer, Meni Wanunu, Will Morrison, Heiko Kuhn, Maxim Frank-Kamenetskii, and Amit Meller.” Nanopore based sequence specific detection of duplex DNA for genomic profiling.” Nano letters 10, no. 2 (2010): 738-742.).

It is to be understood that while the invention has been described in conjunction with the above embodiments, the foregoing description and examples are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains. 

1. A method for detecting a target molecule or particle suspected to be present in a sample, comprising: contacting the sample with (i) a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain, and (ii) a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding, under conditions that allow the target molecule or particle to bind to the ligand and the binding domain to bind to the binding motif; loading the polymer into a device comprising a pore that separates an interior space of the device into two volumes, and configuring the device to pass the polymer through the pore from one volume to the other volume, wherein the device comprises a sensor configured to identify objects passing through the pore; and determining, with the sensor, whether the fusion molecule bound to the binding motif is bound to the target molecule or particle, thereby detecting the presence of the target molecule or particle in the sample.
 2. The method of claim 1, wherein the target molecule is selected from the group consisting of a protein, a peptide, a nucleic acid, a chemical compound, an ion, and an element.
 3. The method of claim 1, wherein the target particle is selected from the group consisting of a protein complex or aggregate, a protein/nucleic acid complex, a fragmented or fully assembled virus, a bacterium, a cell, and a cellular aggregate.
 4. The method of claim 1, wherein step (a) is performed prior to step (b).
 5. The method of claim 1, wherein step (b) is performed prior to step (a).
 6. The method of claim 1, further comprising applying a condition suspected to alter the binding between the target molecule or particle and the ligand, and carrying out the determination again.
 7. The method of claim 6, wherein the condition is selected from the group consisting of removing the target molecule or particle from the sample, adding an agent that competes with the target molecule or particle, or the ligand for binding, and changing the pH, salt, or temperature.
 8. The method of claim 1, wherein the binding motif comprises a chemical modification for binding to the binding domain.
 9. The method of claim 8, wherein the chemical modification is selected from the group consisting of acetylation, methylation, summolation, glycosylation, phosphorylation, and oxidation.
 10. The method of claim 1, wherein the polymer is at least one of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a peptide nucleic acid (PNA), a DNA/RNA hybrid, and a polypeptide.
 11. The method of claim 1, wherein the binding domain is selected from the group consisting of a helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix turn helix, a helix-loop-helix, and a high mobility group box (HMG-box).
 12. The method of claim 1, wherein the binding domain is selected from the group consisting of locked nucleic acids (LNAs), peptide nucleic acids (PNAs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPRs), dendrimers, peptides, and aptamers.
 13. The method of claim 1, wherein the ligand is selected from the group consisting of an antibody, an antibody fragment, an epitope, a hormone, a neurotransmitter, a cytokine, a growth factor, a cell recognition molecule, a nucleic acid, a peptide, an aptamer, and a receptor.
 14. The method of claim 1, wherein the binding domain and the ligand are linked via an interaction selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a metallic bond, a van der Walls force, a hydrophobic interaction, and a planar stacking interaction, or are translated as a continuous polypeptide, to form the fusion molecule.
 15. The method of claim 1, further comprising contacting the sample with a detectable label capable of binding to the target molecule or particle, or the target molecule or particle/ligand complex.
 16. The method of claim 1, wherein the polymer comprises at least two units of the binding motif.
 17. The method of claim 1, wherein the polymer comprises at least two different binding motifs; the sample is in contact with at least two fusion molecules each comprising a different binding domain capable of binding to a different one of the at least two different binding motifs and a different ligand capable of binding to a different target molecule or particle; and the sensor is configured to identify whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.
 18. The method of claim 1, wherein the sensor comprises electrodes further configured to apply a voltage differential between the two volumes and measure current flow through the pore.
 19. The method of claim 1, wherein the device comprises an upper chamber, a middle chamber and a lower chamber, wherein the upper chamber is in communication with the middle chamber through a first pore, and the middle chamber is in communication with the lower chamber through a second pore; wherein the first pore and second pore are about 1 nm to about 100 nm in diameter, and are about 10 nm to about 1000 nm apart from each other; and wherein each of the chambers comprises an electrode for connecting to a power supply.
 20. The method of claim 1, further comprising moving the polymer in a reverse direction after the binding motif passes through the pore, such as to identify, again, whether the fusion molecule bound to each binding motif is bound to a target molecule or particle.
 21. A kit, package or mixture for detecting the presence of a target molecule or particle, comprising: a fusion molecule comprising a ligand capable of binding to the target molecule or particle and a binding domain; a polymer scaffold comprising at least one binding motif to which the binding domain is capable of binding; and a device comprising a pore that separates an interior space of the device into two volumes, wherein the device is configured to allow the polymer to pass through the pore from one volume to the other volume, and wherein the device further comprises a sensor adjacent to the pore configured to identify whether the binding motif is (i) bound to the fusion molecule while the ligand is bound to the target molecule or particle, (ii) bound to the fusion molecule while the ligand is not bound to the target molecule or particle, or (iii) not bound to the fusion molecule.
 22. The kit, package or mixture of claim 21, further comprising a sample suspected of containing the target molecule or particle.
 23. The kit, package or mixture of claim 22, wherein the sample further comprises a detectable label capable of binding to the target molecule or particle, or the target molecule or particle/ligand complex. 